Estrogens are a class of naturally occurring steroid hormones which are produced in the ovaries and other tissues in the body and which directly influence the growth and function of specific target tissues and organs in humans and animals. These specific tissues and organs include the mammary gland, uterus, pituitary, brain, and liver. Although a variety of naturally occurring and chemically synthesized estrogens have been identified and characterized, perhaps the best known is the endogenous estrogen estradiol-17.beta. (also known as E.sub.2).
Estrogens as a class of hormones mediate their action by binding to an intracellular protein identified as "estrogen receptor" (also termed "ER"). The presence of this intracellular ER both provides and accounts for both cell proliferation and protein synthesis by estrogen dependent cells. The estrogen receptor in the absence of the estrogen hormone is biologically inactive both in vivo and in vitro; and, if the cells or tissues are homogenized and fractionated into cytosol and nuclear fractions, the estrogen receptor is found as a soluble protein in the cytosol. Although the precise interactions remain poorly understood, the generally accepted mechanism of action and sequence of events is believed to be as follows: When an estrogen such as estradiol is introduced to the target cells and tissues, there is specific binding between the estrogen and the ER protein which results in the formation of an estrogen/receptor protein complex. Also, at a time subsequent to hormone binding, a process termed activation and/or transformation ensues which leads to the formation of functional estrogen/hormone receptor complexes having a high affinity for the nuclear components, the DNA, of the target cell. Once the hormone/receptor protein complex is physically formed, it is said to translocate as a complex into the nucleus of the cell where it binds to the chromatin at specific binding sites on the chromosomes and initiates messenger ribonucleic acid (mRNA) transcription. New messenger RNA is then synthesized, chemically modified, and exported from the nucleus into the cytoplasm of the cell where ribosomes translate the mRNA into new proteins. This is the well recognized estrogenic effect on the cell--that is, the initiation of new protein synthesis and concomitant new cell growth/proliferation. The theoretical premise and the generally accepted, though poorly understood, mechanism of action regarding estrogen and estrogen receptor proteins and their interactions are described in greater detail by the following publications which are merely representative of the ongoing investigations in this field. These are: Mester et al., Exp. Cell. Res. 81:447-452 (1973); King and Greene, Nature 307:745-747 (1984); Welshons et al., Nature 307:747-749 (1984); Gorski and Gannon, Annu. Rev. Physiol. 38:425-450 (1976); Gorski et al., Recent Prog. Horm. Res. 24:45-72 (1968); Jordan, V., Pharmacological Reviews 36:245-276 (1984).
In order to truly appreciate the background of the present invention, it is useful to summarize in depth the major details and sequential events believed to be in effect regarding the intracellular protein referred to as "estrogen receptor". In the unbound state, and in the absence of an estrogen, the estrogen receptor protein can be located in vitro within the cytosol and is a single protein composed of 595 amino acids. The molecular weight of ER determined from gel electrophoresis and other physical methods is approximately 67,000 daltons. In soluble systems and under set conditions, the ER protein can be found in various molecular forms which sediment at either 8S, 5S, or 4S values as determined by sucrose density gradient analysis. The 8S form of ER is believed to be the unactivated, untransformed form of the ER protein associated with the unbound, inactive state of estrogen receptor in the absence of estrogens. The 8S ER form is a large molecular weight complex, presumably associated with heat shock proteins, that does not bind efficiently to nuclei or DNA in vitro and is stabilized as a macromolecule by sodium molybdate.
In comparison, the 4S ER protein form is a monomeric protein molecule that can be generated from the 8S form in vitro by treatment with high ionic strength buffers or by increasing salt concentrations (KCl or NaCl). The 4S ER form binds to both nuclei and DNA-cellulose in vitro; it is generally termed the "activated but untransformed" estrogen receptor protein. From the published reports, it appears that the dissociation of the 8S ER form into the 4S form initiates either a major change in the sterochemical conformation of the protein or a direct exposure of the previously hidden DNA binding domain of the molecule.
Alternatively, the 5S form of ER is a dimeric protein molecule which is created by the conversion of the 4S ER protein via a bimolecular reaction which is facilitated by elevated temperatures and/or dilution after KCl activation. The 5S form of ER can be generated in vitro by incubation of either the 8S or 4S forms at 28.degree.-30.degree. C. for 30-45 minutes in the absence of transformation inhibitors. It is generally believed that the 5S form of ER is both "activated and transformed" and therefore is the biologically active entity which binds to the DNA within the nuclei. Moreover, it is also this 5S form which is found associated with the nuclei subsequent to the administration of estradiol in vivo.
It will be appreciated that the present state of knowledge regarding the various forms of estrogen receptor protein have been obtained and characterized via many different investigations employing physical and chemical forms of analysis. Merely representative of these various investigations and reports are the following: Muller et al., Endocrinology 116:337-345 (1985); Muller et al., J. Biol. Chem. 258:11582-11589 (1983); Endocrinology Of The Breast: Basic And Clinical Aspects, Volume 464, Annals Of The New York Academy Of Sciences, pages 202-217, 1986; Parmar et al., J. Steroid Biochem. 31:359-364 (1988); Traish et al., J. Biol. Chem. 255:4068-4072 (1980); and Muller et al., J. Biol. Chem. 257:1295-1300 (1982).
It is essential also to recognize that estrogen receptor protein, particularly from human sources, has been investigated and evaluated in terms of functional domains which provide and are responsible for the characteristic biological and physiological properties individually. The complementary DNA (cDNA) of human estrogen receptor has been cloned which, in turn, has lead directly to the elucidation of the human ER protein primary sequence. Subsequent studies have further defined the various functional domains of human ER protein as comparing six different regions, each of which functionally provides different properties and characteristics. Each of the six functional domains have been designated as a region "A-E" respectively. Regions A and B span the first 180 amino acids within ER proteins and have yet to be assigned a precise function in gene expression, although it is postulated that they are required for full functional activity in certain types of cells or for interaction with specific kinds of genes. Region C, which encompasses the amino acid segment 185-263 of human ER protein, is a critical region for biological activity because it is this amino acid segment which is necessary for binding of the ER protein to genomic DNA to occur. This functional domain and its DNA-binding ability is essential for eliciting the estrogen mediated biological response in vivo. Region D is believed to be the hinge area of the protein with as yet an undefined function. Region E is believed to be the steroid binding domain because this region comprises an amino acid sequence which is generally shared between different classes of receptors for steroids. Region F has yet to be assigned a specific function. It is important to note that regions C and E are said to be conserved among all the steroid receptor family members throughout the different classes--thereby indicating that these specific regions are critical for hormone receptor function generally within steroids as a family. Specific publications describing these investigations, data, and conclusions in greater detail are represented by the following: Kumar et al., Cell 51:941-951 (1987); Hill et al., Cancer Res. 49:145-148 (1989); Greene et al., Nature 320:134-139 ( 1986); and Greene et al., Science 231:1150-1153 (1986).
Overall, it will be noted and appreciated that many investigations of ER protein and the characterization of hormone/ER complexes involve immunological methods and assays. A variety of different polyclonal antisera have been prepared against estrogen receptor protein; and against the nuclear binding estradiol-receptor complex typically identified as "estrophilin" [Raam et al., Mol. Immunol. 18:143-156 (1981); Greene et al., J. Ster. Biochem. 11:333-341 (1979); Greene et al., Proc. Natl. Acad. Sci. USA 74:3681-3685 (1977)]. Similarly, a large variety of monoclonal antibodies against human and animal estrogen receptor proteins and estrophilins have been prepared for many different investigational purposes [Greene et al., Proc. Natl. Acad. Sci. USA 74:3681 (1977); Greene et al., Proc. Natl. Acad. Sci. USA 77:157-161 (1980); Greene et al., Proc. Natl. Acad. Sci. USA 77:5515 (1980); Borgna et al., Biochem. 23:2162-2168 (1984); Fauque et al., J. Biol. Chem. 260:15547-15553 (1985); and Moncharmont et al., Biochemistry 23:3907-3912 (1984)].
The common flaw and recurring problem of these known polyclonal and monoclonal antibodies is their uniform and consistent failure to be site specific. This failure, in turn, produces erroneous empirical results and unreliable information--not only for investigational purposes but also in clinical applications of such antibodies for therapeutic purposes. As a major example, the measurement of estrogen receptors in human breast carcinomas has been the primary tool and favored diagnostic method for choosing between hormonal and cytotoxic chemotherapy when treating breast cancer patients. A variety of different immunoassays employing anti-ER antibodies are presently known and used for this purpose. These are exemplified by the following publications: U.S. Pat. Nos. 4,232,001; 4,293,536; 4,215,102; and 4,711,856. See also European Patent Application Publication No. A2-0129669 published Jan. 2, 1985. Unfortunately, the immunoassays employing conventionally obtained monoclonal antibodies for these measurements have been found to be frequently unreliable and often non-specific. The nature and variety of problems of these unreliable and non-specific monoclonal antibodies are illustrated by the following publications: Raam, S. and D.M. Vrabel, Clin. Chem. 32:1496-1502 (1986); Raam, S. and D.M. Vrabel, Clin. Chem. 34:2053-2057 (1988); Raam, S., Steroids 47:337-340 (1986); and Raam, S., Clin. Chem. 33:1107-1108 (1987). Clearly, therefore, given all the presently known antibodies, assays, and immunological techniques, one still cannot accurately predict which of these estrogen receptor positive tumors will respond to hormonal treatment.
The causes of the present dilemma are in fact two fold: First is the failure of the monoclonal antibodies and polyclonal antisera to be sufficiently site-specific in order to demonstrate the presence of estrogen receptor in its various forms. Second is the failure (in so far as is presently known) to be able to identify functional status of this receptor protein using immunoassay systems. It is now clearly apparent to practitioners and clinicians ordinarily skilled in this art that so long as these insufficiently specific antibodies remain in clinical use, many repetitive failures in the known immunoassay systems will occur; and the ability to identify that proportion of breast cancer patients which would be sensitive and responsive to estrogen hormonal treatment will remain plagued with uncertainty and inaccuracy. For these reasons, the development of site-specific antibodies which could be employed within conventionally known diagnostic immunoassays would therefore be recognized generally as a major advance and fundamental improvement in antibody materials, assay reliability, and therapeutic benefit.